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Engineering protein for X-ray crystallography: the murine major histocompatibility complex class II molecule I-A(d)

Academic Article
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Overview

related to degree

  • Scott, Christopher, Ph.D. in Biology, Scripps Research 1993 - 1998

authors

  • Scott, Christopher
  • Garcia, K. C.
  • Stura, E. A.
  • Peterson, P. A.
  • Wilson, Ian
  • Teyton, Luc

publication date

  • February 1998

journal

  • Protein Science  Journal

abstract

  • Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-Adbeta chain. This modified I-Ad molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.

subject areas

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Drosophila melanogaster
  • Histocompatibility Antigens Class II
  • Mice
  • Molecular Sequence Data
  • Ovalbumin
  • Peptide Fragments
  • Protein Engineering
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Research

keywords

  • Drosophila melanogaster protein expression
  • His-tagged protein
  • X-ray crystallography
  • preparative IEF
  • protein deglycosylation
  • surface plasmon resonance
  • thrombin digestion
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Identity

PubMed Central ID

  • PMC2143914

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1002/pro.5560070222

PubMed ID

  • 9521118
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Additional Document Info

start page

  • 413

end page

  • 418

volume

  • 7

issue

  • 2

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